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Ribonuclease II (RNase II) is a bacterial enzyme responsible for 90% of the exonucleolytic degradation of mRNA in bacteria, and has bacterial homologues known to be involved in virulence. The goal of this project was to examine the structural dynamics of RNase II using fluorescence. Prior to the beginning of this project, little was known regarding the structural composition of RNase II – required information in the study of structural dynamics. Consequently, the structure of RNase II was studied by constructing a series of deletion mutants in order to map the domains. The publication of an atomic resolution structure of RNase II allowed the project to move directly into the study of RNase II structural dynamics as it degrades mRNA. As a step towards this, RNase II was fluorescently labeled, and preliminary binding studies of DNA – a competitive inhibitor – to RNase II using fluorescence were conducted. / xii, 90 leaves : ill. (some col.) ; 29 cm

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Chemistry

There is no abstract available for this dissertation. / School of Physical Education, Sport, and Exercise Science

The regulatory enzyme 6-phosphofructo-l-kinase (PFK) controls the key, rate-limiting step in glycolysis. There are 3 known mammalian isoforms termed PFK-muscle (PFK-A), PFK-liver (PFK-B), and PFK-brain (PFK-C) that randomly aggregate to form active homo- and heterotetrameric isozymes with their respective frequencies and kinetic properties contingent upon the presence and concentration of individual subunits. This study utilized RT-PCR and densitometry analyses to characterize the expression patterns of the mRNA for each isoform during mouse preimplantation development. PFK-B is increasingly expressed across these stages with a significant increase in PFK-B transcript between 8-cell (0.425 ± 0.158) and morula (0.579 ± 0.197) stages (p < 0.0005). Neither PFK-A nor PFK-C mRNA was detected at any of the preimplantation stages tested. The statistically significant increase in PFK-B corresponded with the known juncture of the switch from the oxidation of maternally supplied pyruvate to a predominant glycolyticmetabolism. Such timing suggested the direct involvement of elevated PFK-B transcription with an increase in glycolysis. / Department of Biology

Messenger ribonucleic acid (mRNA) turns out to be an increasingly important molecule in forensic analysis of biological samples. Because of the specific role of mRNA in all living cells to transfer genetic information from DNA to proteins, mRNA is able to provide cell-specific information and regulate control of gene expression. mRNA analysis performed on an extracted mRNA sample isolated from a biological stain of a crime scene can be used to identify the nature of the tissue(s) comprising the stain. In this research, the effects of a couple of mRNA storage conditions such as cold temperature and ultraviolet light exposure on mRNA integrity from human saliva have been evaluated. Human saliva samples have been sampled and exposed to UV light and freezing temperature (-20°C) for varying lengths of time. Extracted mRNA from each sample has been quantified spectrophotometrically and subjected to real time RT-PCR to evaluate stability and integrity of one of the saliva marker transcripts, KRT13 mRNA, of treated samples compared to untreated samples. The results of this study indicated that UV light and freezing temperature don’t have a significant effect on the integrity of KRT13 mRNA. There is also no apparent correlation between Ct values of treated samples and treating intervals. This research holds important implications for the use of mRNA for applications in forensic science, an area which has not been researched extensively. / Department of Biology

Xylella fastidiosa is an important bacterial plant pathogen that colonizes the xylem of hundreds of plant species. X. fastidiosa cause Pierce's disease in grapevine by occlusion of the xylem by extensive bacterial colonization, extracellular polysaccharides and the formation of a biofilm. These traits are mediated in a cell-density manner by a cell-to-cell signaling system that transduces a diffusible signaling factor (DSF). This dissertation demonstrates that PD1994, PD1617 and RpfG regulate important traits for bacterial virulence such as cell-cell signaling, biofilm formation and cell aggregation. X. fastidiosa strains harboring mutations in pd1994 (which encodes for a defective GGDEF- EAL-domain protein) and in pd1617 (which encodes for a EAL-domain protein) have increased growth rate, increased biofilm formation, increased plant colonization and decreased cell aggregation. Gene expression analysis of the pd1994 mutant strain showed overexpression of rpfF, which is a DSF synthase, suggesting that PD1994 regulates DSF signaling by repressing rpfF expression. Additionally, the pd1994mutant showed overexpression of pd1617 and rpfG (with EAL and HD-GYP domains respectively, that may be responsible for c-di-GMP turnover), which suggested that this mutant may have low c-di-GMP levels and that PD1994 regulates c-di-GMP turnover by repression of RpfG activity and PD1617 gene expression. X. fastidiosa harboring a mutation on rpfG exhibited decreased biofilm formation while it had no effect in growth or cell aggregation. Together, these results suggest that PD1994, PD1617 and RpfG regulate the DSF regulatory network by controlling the turnover of the second messenger c-di-GMP.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 3 uppsatser.

Thesis (Ph.D.)--University of Western Australia, 2006.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.